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Determination of hepatitis C virus (HCV) genotypes has become increasingly important during the last years for prediction of the clinical course and the outcome of antiviral therapy. Therefore, numerous different methods have been developed to enable HCV genotyping. However, many of them are very laborious and expensive, leading to limited usage in daily routine diagnostics. We have established a method which combines the speed of the new LightCycler technology with the use of amplification products generated for diagnostic quantitative HCV RNA determination. Differentiation of HCV genotypes is performed with these amplicons in a single step by using fluorophore-labeled hybridization probes. Although currently only two different acceptor fluorophores are available for the LightCycler, types 1, 2, 3, and 4, which are by far the prevailing HCV genotypes in Europe and the United States, can be distinguished. Genotypes of specimens from 190 chronically HCV-infected patients were determined by the LightCycler method and compared with the results of nucleotide sequencing. Concordant results were obtained for all samples. This new method offers a fast and convenient possibility to determine the quantitative HCV RNA load and the genotype in large-scale settings within about 4 h.

Objective: Various laboratory methods for detection of cytomegalovirus (CMV) are available, but all suffer from limited sensitivity and specificity or are prone to sampling errors.

The reverse transcriptase V207I mutation within the hepatitis B virus (HBV) polymerase is associated with resistance to lamivudine in vitro. The prevalence of this mutation in treatment-naïve patients was 1% (1/96). A follow-up of the patient carrying this mutation prior to treatment revealed no loss of sensitivity of HBV to lamivudine in vivo.

Human cytomegalovirus (HCMV) is a major pathogen in newborn children and immunosuppressed individuals. There are various diagnostic methods for HCMV, but all test systems either suffer from limited sensitivity and specificity or are prone to sampling errors. Due to its unequaled sensitivity, strong hopes were pinned on the polymerase chain reaction (PCR) for the early diagnosis of active HCMV infections. However, when monitoring immunocompromised patients, unwanted positive results are often obtained without further evidence of active HCMV infection. To better discriminate between latent and active infections, different methods for the quantitation of viral genomes in clinical samples by PCR have been devised in the past years. Here we discuss the current standing of quantitative PCR in HCMV diagnostics.

This study demonstrates the dynamics in the epidemiology of hepatitis C virus subtypes. Subtypes 3a and 4a have become increasingly prevalent in patients where an infection within recent years can be assumed. Evidence is presented that the subtypes observed among younger patients can spread rapidly and lead to significant changes in the subtype distribution.

The serodiagnosis of hepatitis C virus (HCV) infection was analyzed by a recombinant immunoblot assay (RIBA) with recombinant proteins encoded by the viral RNA isolated from our patients in Hamburg, Germany. The HCV RNA was amplified by PCR, and proteins encoded by the viral core and the NS3, NS4, and NS5 regions were expressed subsequently in Escherichia coli. The results obtained with our UKE RIBA were compared with the results of the Abbott HCV second-generation enzyme immunoassay (EIA). Serum samples from 270 patients, which were sent to us on the suspicion of HCV hepatitis and which were negative for hepatitis A virus and hepatitis B virus antibodies, were examined. In 227 cases (84.1%), there were identical positive (204 cases, 75.6%) or negative (23 cases, 8.5%) results in both tests. In 32 cases (11.9%), the reactive Abbott second-generation HCV EIA results could not be confirmed by the UKE RIBA and the HCV PCR. In follow-up studies conducted over 1 year, these results did not change. In three cases (1.1%), the UKE RIBA presented a positive result while the Abbott second-generation HCV EIA was negative. All three cases were positive in the HCV PCR and showed seroconversion in an HCV EIA 4 to 6 weeks later. In addition, 33 patient serum samples were examined by UKE RIBA in parallel with the Ortho RIBA 2.0. In three cases (9.1%), a positive Ortho RIBA 2.0 result could not be confirmed by the UKE RIBA and the HCV PCR. All three patients were free of complaints. The UKE RIBA showed also a smaller number of indeterminate results (3.0%) than the Ortho RIBA 2.0 (24.2%). This comparison study demonstrates that the commercially available HCV antibody tests should be further improved.

The prevalence of hepatitis G virus (HGV) and hepatitis C virus (HCV) infection was determined by reverse transcription-PCR in 777 individuals with and without risk factors for viral transmission via blood. From our results we conclude that transmission of HGV and that of HCV are favored by similar risk factors.

For up to 20 to 40% of patients chronically infected with hepatitis C virus (HCV), the mode of transmission is still unknown. We demonstrate that tear fluid contains HCV RNA-carrying material with the properties of infectious virus and conclude that smear infection with tear fluid may play a role in HCV transmission.

Tandem repeat amplification of resistance determinants in Haemophilus influenzae plasmids is associated with the occurrence of separate circular DNA molecules. They were demonstrated to represent mono- and multimeric forms of the amplifiable segments of the plasmids which comprise the respective resistance transposons and an additional region designated as an amplification sequence. The latter region mediates the recombinational events involved in amplification. The DNA circles apparently lack the ability to replicate autonomously but most probably provide an effective means for the translocation of resistance genes from one plasmid to another.

Summary: The tetracycline-resistant Haemophilus influenzae strains lu121 and fr16017, recently isolated in West Germany, each harbour a plasmid; that of the former (pLU121) has a mol. wt of 31.5 x 106 and that of the latter (pFR16017) has a mol. wt of 33 x 106. Conjugation and DNA-DNA hybridization studies have shown that both plasmids are self-transmissible and carry tetracycline-resistance genes. The purified plasmid DNA of H. influenzae strain lu121 transformed a sensitive Escherichia coli strain to tetracycline resistance. The two R factors are closely related to the H. influenzae plasmid specifying ampicillin resistance (pKRE5367). Electron microscope DNA heteroduplex analysis indicated that pLU121 and pFR16017 probably carry the tetracycline-resistance transposon TnD and that pKRE5367 probably carries the ampicillin-resistance transposon TnA. There is more than one integration site for the insertion which probably represents TnD in pFR16017. All three plasmids have a similar plasmid core and could have a common evolutionary origin.

By means of an ELISA the level of hepatitis B e-antigen (HBeAg) titre was determined in 51 patients with acute and 123 patients with persistent hepatitis B virus (HBV) infection. We also investigated 120 sera of these patients for infectivity by detecting HBV-specific DNA polymerase (DNA-P). Additional determination of the HBeAg titre revealed that the detection of HBeAg at a dilution of at least 1:50 correlates with the detection of DNA-P in 95% of cases, while in sera with an HBeAg titre of 1:25 or less DNA-P is only detectable in up to 5% of cases. Since the detection of DNA-P indicates high infectivity, certain titres of HBeAg can be employed to differentiate three different degrees of infectivity: (1) Sera without HBeAg show no or only little infectivity; (2) sera containing HBeAg≧1:50 are highly infectious; (3) HBeAg titres up to 1:25 indicate a medium degree of infectivity. These three markers for different degrees of infectivity were applied to study the course of infectivity during acute and persistent forms of HBV infection. In acute hepatitis B the period for the elimination of HBeAg is independent of the HBeAg titre in the first serum sample. In all cases the elimination of HBeAg in acute hepatitis B did not exceed 4 months. Patients with persistent HBV infection and an initial HBeAg titre ≧1:50 remained at this high degree of infectivity for at least 18–30 months in 70% of cases. Only 30% of this collective showed a decrease in infectivity. However, 40% of the patients with persistent hepatitis B with an initial low titre of HBeAg showed an increase in infectivity, while 60% remained low or converted to anti-HBe. This confirms that an initially high degree of infectivity in the persistent form of hepatitis B is an unfavourable sign. Also, the present study demonstrates that the correlation of the amount of Dane particles and HBeAg titre is not due to conversion of spontaneously released hepatitis B core antigen (HBcAg) from circulating Dane particles to HBeAg. By means of the ELISA the half-life of HBeAg in vitro was determined to be 12–24 h.

The ampicillin-resistant Haemophilus influenzae strain Ve445 which caused purulent meningitis and septicaemia in a newborn child in Germany contained a 4.4 megadalton (Mdal) plasmid (pVe445) and produced a TEM type beta-lactamase. The transformation to ampicillin resistance of a sensitive Escherichia coli strain with isolated pVe445 DNA proved that the structural gene for the beta-lactamase resided on this plasmid genome. Molecular DNA-DNA hybridization studies and electron microscope DNA heteroduplex analysis indicated that pVe445 probably contained 38 to 41% of the ampicillin translocation DNA segment (TnA) found on R factors of enteric origin. The TnA fragment present in pVe445 most likely does not contain both of the inverted repeat sequences of TnA. DNA-DNA polynucleotide sequence studies indicated that the 4.4 Mdal plasmid pVe445 was unrelated to the 30 to 38 Mdal H. influenzae R plasmids but was closely related to the 4.1 Mdal ampicillin resistance specifying H. influenzae plasmid RSF0885 isolated in the U.S.A. The H. influenzae plasmid pVe445 shared 91% of its base sequences with the beta-lactamase specifying Neisseria gonorrhoeae plasmid pMR0360 (4.4 Mdal) and had 85% of its base sequences in common with the beta-lactamase specifying N. gonorrhoeae plasmid pMR0200 (3.2 Mdal). All of the four 3.2 to 4.4 Mdal beta-lactamase specifying R plasmids of H. influenzae and N. gonorrhoeae investigated probably have a common evolutionary origin.

Sudden and unexpected death and violent death of persons with a high risk of acquiring HIV-infections, especially homosexual males and intravenous drug abusers, have to be investigated by forensic autopsies. Therefore every forensic pathologist has to be aware of this infection and should try to make the proper diagnosis. Three typical cases are described: (1) suicide by hanging, (2) homicide by cutting the throat and (3) intravenous heroin overdose. Merely retro-spectively it could be cleared up that the deceased were homosexual but did not manifestly suffer from AIDS. The morphological findings in the lymph nodes and the postmortem serological findings are described in detail.

An enzyme-linked immunosorbent assay (ELISA) using a highly purified soluble antigen prepared from pathogenic Treponema pallidum (Nichols) was carried out to detect specific IgM antibodies in syphilis. Serum specimens from 365 patients with untreated or treated syphilis were tested by the ELISA and by the 19S(IgM) fluorescent treponemal antibody-absorbed test. In the ELISA sera were preabsorbed with aggregated human IgG to eliminate any rheumatoid factor present. Sera from 331 healthy blood donors served as controls. The results of both tests correlated well. The specificity and sensitivity of the treponemal ELISA were both greater than 97%. The assay was easy to perform and may be automated.

The simultaneous detection test gave no evidence for the presence of RNA tumour viruses in herpesvirus induced malignant lymphomas of non-human primates. The 12 tumours tested were obtained from three different monkey species inoculated with Herpesvirus saimiri or herpesvirus ateles. Particles encapsulating RNA-instructed DNA polymerase and high mol. wt. virus-related RNA were easily demonstrated in tumours of the mouse induced by type-C or type-B oncornaviruses and in human lymphoid cells infected with simian sarcoma virus type I which were examined in parallel. Attempts to demonstrate partial expression of an oncornavirus genome in the herpesvirus induced tumours and attempts to detect an interspecies antigen related to monkey oncornaviruses were negative and strengthened the observations made with the simultaneous detection test.

Intramolecular amplification produces tandem repeats of tetracycline and combined tetracycline-chloramphenicol resistance determinants in conjugative plasmids of Haemophilus influenzae. This process depends on host recombination pathways. Physical mapping revealed the tetracycline transposon involved in amplification to be almost identical with Tn10, including two IS10 insertion elements. The chloramphenicol resistance determinant of the combined transposon is 1.9 kilobases (kb) in size and is bound by two 1.3-kb inverted repeats. Insertion in the close vicinity of the inside end of the left-hand IS10 generates a deletion of a 1.6-kb Tn10 region. The amplifiable units were resolved to comprise not only the respective resistance transposons, but also an additional 1.6-kb sequence (designated AS) which was demonstrated to be identically present in the different amplification systems studied. AS separates amplified transposons from each other, thereby maintaining the same orientation. Moreover, AS is present at the left flank of the transposons, but is missing at the right one. It was shown that AS represents a general constituent of the H. influenzae plasmids of the 45-kb class. Evaluation of the results suggests that AS is responsible for the recombinational events involved in the gene amplification process.

HERPESVIRUSES are suspected to be causal agents in human malignant neoplasia. At the present level of knowledge, clinical trials in man with viral vaccines against oncogenic herpesviruses cannot be initiated, but the oncogenic herpesviruses derived from monkeys1,2 can be used in vivo to study the problems of herpesvirus cancer vaccines. The oncogenic herpesvirus saimiri (HVS) regularly induces malignant lymphoma in cotton topped (CT) marmoset monkeys (S. oedipus). A killed vaccine from HVS was developed in our laboratory and tested for safety and efficacy in this primate model system.

The 36-megadalton Haemophilus influenzae R plasmid pHK539 was found to specify resistance to tetracycline (Tc) and ampicillin (Ap). It was shown by molecular hybridization studies and by electron microscopy that the plasmid pHK539 contained the tetracycline translocation deoxyribonucleic acid (DNA)segment (TnTc) as well as the ampicillin translocation segment (TnAp). The TnAp was integrated in the stem of TnTc. The 34-megadalton H. influenzae R plasmid pRI234 carried a translocatable DNA segment which specified both tetracycline and chloramphenicol (Cm) resistance. Self-annealing and DNA-DNA heteroduplex experiments indicated that this transposon is probably composed of TnTc containing an insertion of a chloramphenicol resistance transposon (TnCm). TnCm is inserted into one of the components of the TnTc inverted repetitions and is itself flanked on both sides by long inverted repetitions. The H. influenzae plasmids pHK539 and pRI234 had more than 60% of their polynucleotide sequences in common with all the other 30- to 40-megadalton R factors recently found in H. influenzae isolates from different countries. The tetracycline-chloramphenicol resistance transposon of pRI234 was integrated twice at different sites in the plasmid after its growth in medium containing tetracycline. The presence of the two copies of the transposon was correlated with higher minimum inhibitory concentrations against tetracycline as well as against chloramphenicol. After its growth in medium containing tetracycline, the H. influenzae R plasmid pFR16017 specifying Tc resistance contained one, two, three, or even four copies of TnTc integrated at different sites in the plasmid, or the loop of TnTc was amplified. The heterogeneity of the pFR16017 plasmid was seen in all single-colony isolates and correlated with a higher minimum inhibitory concentration against tetracycline.

The ampicillin-resistant isolate Haemophilus influenzae KRE5367 which produced a TEM type β-lactamase contained a 30 megadalton (Mdal) plasmid (pKRE5367) with a copy number of approximately 2 per chromosomal equivalent and a mole fraction guanine plus cytosine content of 0.39. Ampicillin resistance was transferred by cell-to-cell contact, and the transformation to ampicillin resistance of a sensitive Escherichia coli strain with isolated pKRE5367 DNA proved that the structural gene for the TEM type β-lactamase resided on the plasmid genome. It was shown by molecular hybridization that pKRE5367 probably contained the ampicillin translocation DNA segment (TnA) found on some R factors of enteric origin. The H. influenzae plasmid pKRE5367 had most of its polynucleotide sequences in common with other R factors recently isolated from H. influenzae. DNA-DNA hybridization studies indicated that pKRE5367 shared about 70% base sequence homology with the two tetracycline-resistance R plasmids pLU 121 (31.5 Mdal) and pFR 16017 (33 Mdal) recently isolated in W. Germany. In addition, pKRE5367 had nearly 100% of its polynucleotide sequences in common with the 30 Mdal ampicillin-resistance R plasmid RSF007, isolated in the U.S.A.; it shared 70% base sequence homology with the 31.5 Mdal tetracycline-resistance plasmid pUB701, isolated in the U.K.; and it had 65% of its polynucleotide sequences in common with the 38 Mdal tetracycline- and chloramphenicol-resistance R plasmid pR1234 isolated in the Netherlands. Despite the broad geographical range, the R plasmids of H. influenzae appeared to be closely related.

The Haemophilus influenzae R plasmids specifying resistance against one, two, or three antibiotics which have emerged in different parts of the world were shown to have closely related but not identical plasmid cores. The gene for ampicillin resistance in the H. influenzae plasmid pKRE5367 is part of a transposon similar to Tn3, which was transposed from pKRE5367 onto RSF1010 in Escherichia coli. An indigenous H. influenzae plasmid (pW266) was isolated. Its properties correspond to those of the H. influenzae R plasmids, except for the presence of a drug resistance transposon. The in vitro-generated H. influenzae R plasmids carrying an ampicillin resistance transposon, a tetracycline resistance transposon, and a transposon for combined tetracycline-chloramphenicol resistance resembled the natural isolates. The findings support the hypothesis that the R plasmids of H. influenzae are of multiclonal evolutionary origin.

Virulence of Yersinia enterocolitica O:8 is associated with pO:8, a 42-megadalton plasmid. We constructed a mobilizable pO:8 derivative by successive in vitro and in vivo genetic manipulations. The in vitro constructed hybrid molecule pRK290B8-5 consisting of the mobilizable vector pRK290B and a 2.9-megadalton BamHI fragment of pO:8 was conjugally transferred to a Y. enterocolitica strain of serotype O:8 which harbored the virulence plasmid pO:8. From Yersinia transconjugants, a cointegrate was isolated which apparently formed by homologous recombination between the two component plasmids. The cointegrate was mobilized into plasmidless Y. enterocolitica strains of different serotypes. The transconjugants of serotype O:8 were found to express all four plasmid-associated phenotypes: (i) mouse lethality (Ml), (ii) conjunctivitis provocation in the guinea pig eye (Con), (iii) calcium requirement for growth at 37 degrees C (Mox), and (iv) agglutinogens (Ag8). The transconjugants of serotype O:3 expressed the phenotypes Con, Mox, and Ag8 but were nonlethal for mice (Ml-). The transconjugants of serotype O:5 remained avirulent for mice (Ml-) and for the guinea pig eye (Con-) but expressed the phenotypes Mox and Ag8. These data show that the virulence plasmid is probably not functionally interchangeable within different serotypes of Y. enterocolitica.

The periods of persisting immunoglobulin M class antibodies to hepatitis B core antigen in 28 patients with acute hepatitis B infections and in 134 patients with chronic hepatitis B infections were studied by using an enzyme-linked immunosorbent assay and an indirect immunofluorescence technique. We showed that the clearance of antibodies to hepatitis B core antigen is independent of the degree of viremia in both forms of hepatitis.

Mobilizable virulence plasmids of Yersinia enterocolitica of serotypes O:3 and O:9 were constructed by cointegration of a mobilizable vector into the virulence plasmids. The obtained cointegrates were mobilized into plasmidless Y. enterocolitica strains of serotypes O:3, O:5, O:8, and O:9. The transfer experiments revealed the existence of two different subgroups of plasmid-associated traits. (i) Animal virulence functions (mouse lethality and conjuctivitis provocation) were only transferable to plasmid-cured derivatives of virulent parent strains (serotypes O:3, O:8, and O:9), but they were not transferable to Y. enterocolitica antigen reference strains (serotypes O:3 and O:8) or to a plasmidless clinical isolate of serotype O:5. A further striking result was that a serotype O:8 strain regained the mouse lethality trait after receipt of a plasmid from a strain not lethal to mice. These results demonstrate that plasmid-mediated animal virulence functions are not uniformly expressed within Y. enterocolitica. (ii) The second subgroup of plasmid-mediated traits (calcium dependency, surface agglutinogens, HEp-2 cell adherence, and protein release) were transferable to all Y. enterocolitica recipient strains tested (serotypes O:3, O:5, O:8, and O:9 of different origin). For the first time HEp-2 cell adherence and temperature-induced release of five major protein species are described as transferable traits.

A double immunofluorescence staining technique is described for differentiation between cell-attached (extracellular) and ingested (intracellular) bacteria by HEp-2 cells in cell culture monolayers. This method is based upon the observation that membranes of viable mammalian cells are impermeable for antibodies but are rendered permeable by treatment with fixatives. Consequently, extracellular bacteria can be stained by specific rhodamine-labeled antibodies before fixation, and intracellular bacteria can be visualized by treatment with specific fluorescein-labeled antibodies after fixation. The accuracy and simplicity of this method is demonstrated with HEp-2 cell culture monolayers as target cells and an isogenic pair of Yersinia enterocolitica, one of which is phagocytosis resistant and the other of which is phagocytosis sensitive. Furthermore, it is shown that this staining technique is also applicable for studying the interaction of bacteria with macrophages and fibroblasts.

The association of Campylobacter pyloridis with antrum gastritis and peptic ulcers was described. We investigated antral biopsies from 180 patients who underwent gastroscopy. By culture or Gram stain or both, we found overall 98 (54%) of them to be positive for C. pyloridis. In the various groups the following percentages were found to be positive: normal antral mucosa 3% (n = 30); moderate superficial antrum gastritis, 49% (n = 83); severe superficial antrum gastritis, 86% (n = 44); duodenal ulcer, 83% (n = 54); and gastric ulcer, 72% (n = 18). A serological screening that used a complement fixation test yielded the following results: highest rates of positive complement fixation titers were seen in patients with severe gastritis and those with duodenal ulcers, both with 79%; the lowest incidence was in a group of 20 blood donors, with 5%. Positive complement fixation titers in gastritis patients also correlated well with characteristic patterns on immunoglobulin G and A immunoblots, while there was no specific reactivity observed on immunoglobulin M immunoblots.

Killed herpesvirus cancer vaccines were prepared by inactivation of oncogenic herpesviruses (Herpesvirus saimiri and Herpesvirus ateles) with heat and formaldehyde. The killed vaccines, which are not free of viral nucleic acid, were safe in 121 vaccinated monkeys of 4 different species. Some of these monkeys have now been under observation for 2 years. The vaccines induced high titers of specific humoral antibodies in all vaccinated monkeys. The killed vaccines proved to be efficient against the i.m. challenge with cell-free oncogenic herpesviruses. The challenged animals remained clinically well without signs of an infection and have now been under observation for 1 year, while the nonvaccinated control monkeys died of malignant lymphoma 34 to 52 days after inoculation. The protection against H. saimiri could be passively transmitted with the serum of the vaccinated monkeys. The vaccination did not prevent but delayed tumor development after tumor cell transplantation. The resistance against the oropharyngeal route of infection remains to be determined.

HERPESVIRUSES are the most important candidates for human cancer agents. The Epstein–Barr virus (EBV) in particular is suspected of being the causal agent in Burkitt's lymphoma and nasopharyngeal carcinoma1. Immunological principles, well understood from vaccine prophylaxis of viral diseases, provide the potential for the eventual control of herpesvirus-induced neoplasia. The development of safe and efficient cancer vaccines with the human candidate herpesviruses is, however, difficult, as there are no reliable in vitro or experimental animal methods for detecting oncogenic potential in preparations which might be applicable to the human subject. We are studying the problems of herpesvirus cancer vaccines in non-human primates. Two members of the herpesvirus family, H. saimiri (HVS) and H. ateles (HVA), induce malignant lymphoma and leukemia in several New World monkey species2,3. HVS-induced lymphoma in cotton topped (CT) marmoset monkeys (Saguinus oedipus) has been used in this study as all monkeys inoculated with HVS regularly develop malignant lymphoma within two months4,5. This investigation describes the effect of a killed herpesvirus cancer vaccine in protecting non-human primates against malignant lymphoma.